Photosynthetic characterization of Arabidopsis leaves: diffusional and biochemical aspects
Growing Arabidopsis plants in an ‘ice-cream cone-like’ soil structure (Fig. 1) successfully permitted clamping of its leaves in the 2 cm2 area of the gas exchange and chlorophyll fluorescence chamber of the Li-6400 (Li-6400-40) covering the entire cuvette. Therefore, it was possible to perform simultaneous measurements of gas exchange and chlorophyll fluorescence in Arabidopsis leaves, as it is for other species with larger leaves using the same chamber size. Stomatal conductance (gs) values were similar to those reported for Arabidopsis in the literature (Lascève et al., 1997; Poulson et al., 2002). AN values in young plants also agreed well with the highest values reported in the literature (Walters et al., 2004), although the reported values are often much lower, ranging from 6 to 10 µmol CO2 m−2 s−1 (Lake, 2004). However, many reports do not specify the plant's age at measuring time (Lake, 2004; Walters et al., 2004; Büssis et al., 2006). Values reported for whole-plant measurements are usually much lower (Dodd et al., 2004; Lake, 2004; Tocquin & Perilleux, 2004), and this may be the result of a combination of different leaf ages and leaf superimposition leading to decreased light interception.
To the best of our knowledge, no previous report of mesophyll conductance to CO2 (gm) was available for Arabidopsis. The present results show that maximum gm was approx. 0.2 mol CO2 m−2 s−1 bar−1, and it decreased substantially with plant age. These data were confirmed using three well-accepted independent methods: the gas exchange-chlorophyll fluorescence method by Harley et al. (1992), the curve-fitting method by Ethier & Livingston (2004), and the online carbon isotope discrimination method by Evans et al. (1986). As is usually observed (Evans & Loreto, 2000), gm was well correlated with both AN and gs (Fig. 3). The relationship between AN and gm found in Arabidopsis did not extrapolate to zero, and it significantly deviated from that described by Evans & Loreto (2000) for several species. Such a deviation and nonzero intercept have already been shown in some species (Singsaas et al., 2003), and are thought to occur because the relationship is actually curvilinear, deviating from linearity particularly at gm values below 0.05 mol CO2 m−2 s−1 bar−1 (Warren & Adams, 2006). The relationship between gm and gs substantially deviated from 1 : 1, as has been found in some studies (Ethier et al., 2006) but not in others (Loreto et al., 1992). The slope of this relationship suggests that there is a shift in photosynthesis limitation from predominantly gs, when gs is higher than 0.18 mol CO2 m−2 s−1, to predominantly gm, when gs drops below 0.18 mol CO2 m−2 s−1.
While gm values found in Arabidopsis corresponded well with observed AN and gs, they were much lower than expected for a species with very thin leaves (leaf mass per area (LMA) was only 18 g m−2 in the studied plants, with no significant differences between ages). For instance, similar values of gm (c. 0.20–0.25 mol CO2 m−2 s−1 bar−1) have been described for species with thicker leaves, such as Vitis vinifera (Flexas et al., 2002; LMA = 65 g m−2), Citrus paradisi (Syvertsen et al., 1995; LMA = 108 g m−2), or even Pseudotsuga menziesii (Warren et al., 2003; LMA = 150–200 g m−2). On the contrary, much larger gm values (0.3–0.6 mol CO2 m−2 s−1 bar−1) are usually described for species having similar LMA to Arabidopsis (i.e. < 50 g m−2), such as Prunus persica (Syvertsen et al. (1995), Nicotiana tabaccum (Evans & Loreto, 2000; Flexas et al., 2006) and Phaseolus vulgaris (Singsaas et al., 2003). Therefore, we suggest that gm is limiting photosynthesis in Arabidopsis plants, at least when growing under the standard laboratory conditions used here and in most studies with this species. This could be reflecting low-light acclimation (Piel et al., 2002), although relatively low gm in thin leaves has also been observed in sun leaves of several Mediterranean herbs (Galmés et al., in press). The recent discovery that aquaporins are strongly implicated in the regulation of gm (Hanba et al., 2004; Flexas et al., 2006) provides a strong physiological background to explain the weakness of the relationship between gm and leaf structure.
The AN–Ci curves and derived parameters determined in young plants were very similar to those already shown for Arabidopsis at the leaf level by Walters et al. (2004) and Büssis et al. (2006), showing a maximum AN of c. 20 µmol CO2 m−2 s−1, attained at a Ci of 600–1000 µmol CO2 mol−1 air, and a photosynthesis decline associated with restricted TPU at higher Ci. When measured at the whole-plant level, however, the AN–Ci curves are quite different, with lower initial slopes and not showing CO2 saturation even at Ci values above 1200 µmol CO2 mol−1 air (Poulson et al., 2002; Tocquin & Perilleux, 2004). In fact, the only Vc,max_Ci values found in the literature are those by Tocquin & Perilleux (2004), determined at the whole-plant level and ranging between 21 and 45 µmol m−2 s−1, that is, substantially lower than those found in the present study. The values observed in the present study in Arabidopsis for Vc,max_Ci, Jmax_Ci and VTPU_Ci are well within the range of those typically found in the literature, and the ratios Jmax_Ci/Vc,max_Ci were very similar to those usually found in C3 plants at 25°C (Wullschleger, 1993). However, absolute rates of Vc,max_Ci, Jmax_Ci and VTPU_Ci in Arabidopsis are near the lower end of the range found in the literature, and more similar to those found in leaves adapted to low light (Piel et al., 2002), with low nutrient supply (Warren, 2004) or thick-leaved sclerophyll species (Wullschleger, 1993), than to those generally observed for herbaceous dicots (Wullschleger, 1993). These data, combined with gm, suggest that a low carboxylation capacity might also limit maximum photosynthesis in Arabidopsis.
However, it has been shown that AN–Ci curves underestimate Jmax and, especially, Vc,max, leading to some overestimation of the ratio Jmax : Vc,max (Flexas et al., 2002, 2006; Piel et al., 2002; Warren, 2004). The need to use estimates of chloroplastic CO2 concentration for a better photosynthetic characterization of Arabidopsis has recently been highlighted (Tocquin & Perilleux, 2004). The AN–Cc curves determined in the present study show that Jmax and Vc,max were only slightly, although nonsignificantly, underestimated by AN–Ci curves in young plants, but substantially underestimated in old plants, that is, those with lower gm. The ratio Jmax_Cc : Vc,max_Cc was actually lower than the ratio Jmax_Ci : Vc,max_Ci (Table 1). It may be argued that the estimation of these parameters are strongly affected by the choice of kinetic parameters involved in the equations (Kc, Ko and their dependence on temperature). However, since these parameters have not yet been described for Arabidopsis, any choice of parameters published for other species could be considered arbitrary. Consequently, we have selected the Ci-based and Cc-based parameters described by Bernacchi et al. (2001, 2002) for tobacco because, as both were calculated for the same species, a comparison between Ci-based and Cc-based photosynthesis parameterization would be straightforward. Hence, while there might be some uncertainty in the absolute values of Vc,max and Jmax, at least the differences between Ci-based and Cc-based values may be regarded as quite approximate. Therefore, the present results demonstrate that parameters derived from CO2-response curves of photosynthesis are underestimated when Ci is used instead of Cc, and that proper AN–Cc may be necessary to accurately analyse photosynthetic limitations imposed by plant ageing (see the following section) or environmental stresses.
Photosynthesis decline with plant age in Arabidopsis: diffusional and biochemical limitations
Net photosynthesis was depressed by as much as 40% from 30- to 40-d-old plants. Age-dependent decreases in photosynthesis of a similar magnitude over similar time ranges have been characterized in some herbs, such as Triticum durum (Loreto et al., 1994), Spinacia oleracea (Delfine et al., 1999) and Nicotiana sylvestris (Priault et al., 2007), but not in Arabidopsis. In perennial deciduous or evergreen plants, similar decreases are observed but over much longer time periods, such as months to years (Grassi & Magnani, 2005; Niinemets et al., 2005; Ethier et al., 2006).
It has recently been demonstrated that, in evergreen trees, most of the age-related decline in photosynthetic capacity in old leaves is the result of reduced gm and, to some extent, reductions in the activation state but not in the amount of Rubisco (Niinemets et al., 2005; Ethier et al., 2006). In deciduous perennials, by contrast, most of the age-related decline in photosynthetic capacity in autumn is the result of reduced photosynthetic biochemistry, with only a minor contribution of reduced gm (Grassi & Magnani, 2005). In annual plants, despite decreased gm in older leaves, it is usually assumed that age-related photosynthetic decline is mostly associated with the degradation of chlorophylls and the breakdown of Rubisco and other chloroplast proteins into amino acids (Friedrich & Huffaker, 1980; Ethier et al., 2006). However, Loreto et al. (1994) suggested that the age-related decline in photosynthesis in wheat may be attributed in part to a reduction in gm and in part to a decline in the amount of Rubisco. On the other hand, Delfine et al. (1999) showed in spinach that a 40% decline in photosynthesis of 50-d-old vs 22-d-old leaves was associated with a nonsignificant change in Rubisco amount and activity, a 25% decline in gs and a decline in gm of as much as 70%. However, a quantitative photosynthesis limitation analysis was lacking in these studies, which may be necessary to establish the physiological basis for age-related decline in photosynthesis in annual plants.
Here we present the first quantitative photosynthesis limitation analysis in response to leaf ageing performed in Arabidopsis and, to the best of our knowledge, in any annual species. The results clearly show that a 41% decline in photosynthesis as a result of leaf ageing was entirely caused by increased diffusional limitations to CO2 transfer, and not to biochemical restrictions. The largest limitation (28%) was the result of reduced mesophyll conductance to CO2, while decreased stomatal conductance accounted for the remaining 13%. The present results show, for the first time, that, similar to evergreen perennials, age-related photosynthesis decline in the annual plant A. thaliana is initiated by decreased mesophyll conductance to CO2. Chlorophyll and protein degradation may appear at a later stage, when photosynthesis is already largely depressed.